Improved outcomes for patients with breast cancer is dependent on both the earlier detection of malignant lesions when local therapy is the most effective, and a marked improvement in our current approaches to curing patients with metastatic disease. While biological response modifiers such as trastuzumab and bevicizumab have improved response rate and time to progression of patients with metastatic breast cancer, the long-term outcome remains quite poor, with fewer than 2% of patients alive 10 years after diagnosis. Immunotherapy offers a new approach to the treatment of patients with metastatic breast cancer. Previous studies have shown that tumor cells that are resistant to chemotherapy can be effectively killed by cytotoxic T cells. Additionally, immunotherapy offers the possibility of immune memory allowing therapy in the past to remain viable into the future. Despite these benefits, immunotherapy in the treatment of patients with solid tumors outside of malignant melanoma has been disappointing. Clinical complete responses are not seen, memory does not typically persist post vaccination, and vaccines can induce T cells without effector function. Furthermore, the induction of anti-tumor responses to self-antigens may induce regulatory T cells (Treg) and myeloid suppressor cells that block the function of our current vaccines. Thus, new approaches to tumor immunotherapy need to focus on the induction of robust immune responses that include antigen-specific CD4+ and CD8+ T cells, antibody formation and blocking the action of regulatory mechanisms. A critical flaw in active vaccination is the lack of production of proinflammatory cytokines post vaccination critical for T cell polarization and inhibiting Tr9g function. Our group has focused on the use of viral vectors with a specific tropism for dendritic cells (DCs) that induce robust proinflammatory cytokines. We have found that Venezuelan equine encephalitis replicon particles (VRP) encoding specific antigens induce more robust T cell response in vitro compared to the use of peptide-pulsed DCs. Moreover these DCs are capable of generating significant quantities of proinflammatory cytokines in vitro and enhanced T cell response in vivo. In this proposal, we will evaluate the activity of mouse DCs infected with VRP encoding HER-2/neu in breaking tolerance and inducing tumor remission in FVB-Neu transgenic mice that are tolerant to tumors expressing Neu. In aim 1, we will investigate whether this platform leads to enhanced survival of FVB-Neu mice that have had tumors implanted prior to vaccination. The role of type I interferons will be evaluated and whether this vaccine can induce antigen-specific CD4+ T cells assessed. Additional experiments will determine if vaccine efficacy is enhanced by blocking the function of myeloid suppressor or Treg cells using specific chemotherapy. In aim 2, we will determine if small molecule inhibitors of CCR5 can inhibit Treg trafficking to the tumor site and enhance the efficacy of vaccination. Aim 3 involves a clinical trial evaluating the efficacy of chemotherapy with VRP-Her-2/neu infected DCs in patients with metastatic breast cancer. Finally aim 4 will evaluate whether we can generate T cell responses to the cancer-testis antigens MAGE-A1 and MAGE-A3 in Luminal B subtype tumors. As with our previous SPORE project, our goal is to use our preclinical platform as a springboard to develop novel therapy for patients with metastatic breast cancer.